Tevenvirinae phages encode a family of LSm-like fold proteins.

Uncharacterized bacteriophage proteins typically have little homology outside the phage world. An example is the T4 early protein GoF. Although the function of wild type is not known, the GoF mutant (D25Y) affects the level of T4 gene mRNA under certain conditions. To investigate possible GoF functions, we leveraged the power of AlphaFold3. We found that despite having very dissimilar sequences, GoF and 2 other uncharacterized T4 early proteins, MotB.1 and Frd.2, are structurally similar with predicted N-terminal LSm-like fold motifs. Since this motif, which is found throughout biology, is frequently associated with an RNA function and the GoF(D25Y) mutation is found within the predicted LSm-like fold, we hypothesized that these proteins may affect gene expression. Consequently, we used a fluorescent translational reporter system and RT-qPCR to investigate if and how the presence of the proteins affect the expression of an gene placed downstream of the T4 gene 5' untranslated region. We find that the heterologous expression of (D25Y) increases the level of mCherry post-transcriptionally by increasing the stability of the RNA. However, neither WT GoF nor MotB.1 have this effect. We speculate that GoF(D25Y) may represent a gain-of-function mutant that can increase RNA stability. Using AlphaFold3 models we speculate how the D25Y mutation in GoF might facilitate or enhance RNA binding. Our work reveals the power of AlphaFold to find unexpected structure/function relationships among uncharacterized proteins.